A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [ 18 F]FB-ML5, as a molecular probe for

Scheme 1: Synthesis of the building block 9 Figure 1: Structure and design of ML5

4. Materials and methods 1 General

4.2 Statistical analysis

Calculations were performed using Excel 2007 (Microsoft) and GraphPad prism 5.0 for Windows (GraphPad Software, San Diego, USA). Results are expressed as mean ± SD. Comparisons between different experimental groups were made using unpaired two-sided student t-test. Data were considered statistically significant when p values were smaller than 0.05.

4.3 Molecular modeling of ML5 and FB-ML5

The crystal structures of MMPs and ADAM-17 were downloaded from the Protein Data Bank (PDB) with MMP-2 (PDB code 1HOV), MMP-9 (PDB code 2OW1), MMP-12 (PDB code 1JK3) and ADAM-17 (PDB code 2I47). All molecules were drawn using ChemaxonMarvinSketch (www.chemaxon.com) and prepared (structure recogni-tion and protonarecogni-tion) using SPORES (www.tcd.uni-konstanz.de/research/spores.

php). Molecular docking simulations were performed using PLANTS v1.6.140,141.

The docking site center was determined by applying a constraint for the hydroxamic group to be able to form a coordination with the zinc in the active site. Fifteen poses were generated for each compound and the docking results were analyzed using Molegro Virtual Docker (www.molegro.com). Docking solutions were selected

based on the MOLDOCKSCORE and the docking solutions were evaluated manually, followed by energy minimization of the ligand.

4.4 Synthesis of ML5

The peptidyl MMP inhibitor ML5 [Fig 1] was synthesized following slight modi-fications of the literature procedures [27, 28]. Briefly, the Evan’s template 1 was prepared in a 3-step reaction from the Boc-(D)-phenylalanine. The latter analogue was treated with triethylamine and ethyl chloroformate in order to get the cor-responding acid anhydride which was then reduced with sodium borohydride to give the corresponding alcohol. After treatment with thionyl chloride, the resulting tert-Bu-ester was transformed in its acyl chloride, which was then intramolecu-larly attacked by the primary alcohol to obtain the Evan’s template 1. 4-Methyl-pentanoyl chloride 2 was obtained after treatment of 4-methyl-pentanoic acid with thionyl chloride. N-Boc-O-TBS-hydroxylamine was prepared in a 2-step procedure from the hydroxylamine hydrochloride. The amino group of the latter analogue was protected with di-tert-butyl dicarbonate. The resulting compound was treated with tert-butyl dimethylsilyl chloride to give N-Boc-O-TBS-hydroxylamine. The building block 9 was prepared in 7 steps [Scheme 1], and started by deprotonation of the Evan’s template 1 with n-butyllithium, followed by acylation with 4-methyl-pentanoyl chloride 2 to give 3. Removal of the acidic alpha-proton with lithium bis(trimethylsilyl)amide followed by reaction of the resulting enolate with tert-butyl-bromoacetate gave 4. Treatment of 4 with lithium benzyl alcoholate led to the benzyl ester 5. The tert-butyl-ester 5 was then deprotected to give the intermediate 6. The obtained carboxylic acid 6 was transformed into the corresponding acyl chloride, which was treated with N-Boc-O-TBS-hydroxylamine to give 7. The benzyl ester in 7 was hydrolyzed by catalytic hydrogenation to the acid 8. The carboxylic acid 8, after activation with N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide, was then acylated with pentafluorophenol to give the building block 9. Dipeptide 10 was synthesized on Rink amide resin using standard protocols [Scheme 2]. After deprotection of the Fmoc group, the Fmoc-protected phenylalanine was incorpo-rated. Subsequent removal of the Fmoc was followed by the coupling of the building block 9 under the agency of iPr2EtN. The resulting compound was cleaved from the resin and simultaneously deprotected with 95% aqueous TFA. The obtained compound was purified and characterized by LCMS.

CHAPTER

3

4.5 Synthesis of the acylating agent SFB

Synthesis of SFB by the first approach

To a solution of 4-fluorobenzoic acid (1.0 g, 7.1 mmol) and N-hydroxysuccinimide (0.9 g, 7.8 mmol) in 20 mL of DCM and 1 mL of DMF at 0°C was added dropwise a solution of N,N’-dicyclohexylcarbodiimide (1.6 g, 7.8 mmol) in 10 mL of DCM at 0°C.

The reaction was stirred overnight at room temperature. The reaction was filtered to remove the N,N’-dicyclohexyl urea and the obtained filtrate was concentrated under reduced pressure. The resulting residue was partitioned between EtOAc and water. The organic layer was washed with water (3 times), brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product. The latter was recrystallized from EtOAc-hexane to afford SFB (0.67 g) as a white solid.

Synthesis of SFB by the second approach

To a solution of 4-fluorobenzoic acid (0.1 g, 0.7 mmol) and sodium carbonate (0.07 g, 0.66 mmol) in 3 mL of ACN, stirred for 5 min, was added TSTU (0.21 g, 0.7 mmol).

The mixture was stirred for 1 h at 50°C and was then filtered. The resulting filtrate was diluted with 10 mL 1% acetic acid and was passed through a Lichrolut EN cartridge. The cartridge was washed with 10 mL 0.01% AcOH/ACN (70/30) (v/v) and was dried with a stream of nitrogen. The cartridge was eluted with 3 mL of ACN and subsequent evaporation gave SFB (0.14 g) as a white product.

1H NMR (chloroform-d) δ 8.24 – 8.13 (m, 2H), 7.48 – 7.37 (m, 2H), 2.81 (s, 4H).

4.6 Synthesis of the reference compound FB-ML5

Synthesis of FB-ML5 by SPPS

The SPPS resulting to the reference compound FB-ML5 followed a procedure iden-tical to the synthesis of ML5.

Synthesis of FB-ML5 in solution

The acylation in solution with NHS-coupling agents proceeds generally in borate buffer at pH 8.5. However, it was shown that boronic acid complex can be formed with hydroxamic acid moieties [38]. As ML5 contains a hydroxamic acid, we considered phosphate buffer as a substitute of borate buffer. This buffer was

pre-pared by a Na2HPO4.12H2O 1 M /NaH2PO4.H2O 1 M (93.2/6.8) (v/v) solution and by adjustment of the pH to 8.5 with sodium hydroxide 1 M. ML5 (0.50 mg, 1.08 µmol, 500 µL) dissolved in phosphate buffer pH 8.5 (0.01 M) was transferred to SFB (1.08 µmol, 500 µL) in ACN. The reaction mixture was allowed to react for 30 min at 50

°C. The reaction was quenched with HCl (1 mL, 0.25 M). Then an aliquot of 100 µL was injected through an analytical HPLC, using a Phenomenex Luna C18 column (4.6 mm x 250 mm, 5 μm) from Waters, preceded of a 20 x 4.6 mm2 precolumn.

Gradient elution was performed using a mixture of H2O + 0.1% TFA (solvent A) and CH3CN + 0.1% TFA (solvent B). A linear gradient (overall time = 60 min) starting from 95% solvent A in solvent B to 100% solvent B at 60 min was employed at a flow rate of 1 mL.min-1. The column effluent was monitored using an Elite Lachrom VWR Hitachi L-2400 UV detector (λ = 254 nm, AUFS = 0.010) and a Bicron frisk-tech radioactivity detector. Sample injection was carried out using an injector block with a loop of 100 µL. Fractions of 1 min were collected and the formed products were identified by mass spectrometry.

The retention time of FB-ML5 was 37 min.

ESI-MS (m/z): 586.5 [M+H]+, calc. 586.7; 608.5 [M+ Na]+, calc. 608.7.

Synthesis of FB-ML5 in solution by using an excess of acylating agent

ML5 (0.50 mg, 1.08 µmol, 500 µL) dissolved in phosphate buffer pH 8.5 (0.01 M) was transferred to SFB (5.40 µmol, 500 µL) in ACN. The reaction mixture was al-lowed to react for 30 min at 50 °C. The reaction was quenched with HCl (1 mL, 0.25 M). Then an aliquot of 100 µL was injected through an analytical HPLC, as previously described. Fractions of 1 min were collected and the formed products were identified by mass spectrometry.

The obtained product was 2FB-ML5 with a retention time of 26 min.

ESI-MS (m/z): 708.4 [M+H]+, calc. 708.8; 730.4 [M+ Na]+, calc. 730.8.

4.7 In vitro evaluation of ML5, FB-ML5 and 2FB-ML5 in a fluorogenic

In document University of Groningen Design, (radio)synthesis and applications of radiolabelled matrix metalloproteinase inhibitors for PET Matusiak, Nathalie (Page 80-83)